Reagent composition for biosensor and biosensor having the same

ABSTRACT

This invention discloses a reagent composition for a biosensor having high sensitivity which is capable of improving analysis linearity of an analyte such as glucose by reacting an oxidoreductase, a metal-containing complex, and Naphthol Green B, and minimizing blood necessary for measuring blood glucose since it is possible to detect a concentration of a small amount of the analyte, and a biosensor having the same. The reagent composition for a biosensor includes at least two kinds of electron transfer mediators including Naphthol Green B, and an enzyme.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims priority to Korean Patent Application No.10-2013-0077335, filed on Jul. 2, 2013, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND

1. Field

The present invention relates to a reagent composition for a biosensorhaving high sensitivity, which is capable of improving analysislinearity of an analyte such as glucose, detecting a concentration of asmall amount of analyte, and minimizing blood necessary for measuringblood glucose, and a biosensor having the same.

2. Discussion of Related Art

The present invention relates to a reagent composition for a biosensorand a biosensor having the same, and particularly, to a reagentcomposition for quantifying a specific ingredient of a biological sampleand a biosensor having the same.

Recently, as the number of diabetic patients increases, necessity ofperiodically measuring an amount of blood glucose has been increased inorder to diagnose and prevent diabetes. Such diabetes is known as amajor health risk factor. In general, American Diabetes Association(ADA) recommends that most insulin-dependent diabetic patients check theblood glucose three or more times per day. Insulin controls use of bloodsugar and prevents hyperglycemia that may cause ketosis when diabetes isnot treated. However, inappropriate management of an insulin therapy maycause hypoglycemia. Since hypoglycemia may cause a coma, it may be fatalfor patients.

In addition, when patients suffer from diabetes for a long time,complications such as heart disease, atherosclerosis, blindness, astroke, hypertension, and renal failures may be caused. Since an amountof insulin injection is associated with an amount of the blood glucose,accurate detection of the blood glucose is essential to appropriatelytreat diabetes.

Accordingly, various blood glucose meters are produced such that theblood glucose may be easily measured using portable instruments. Ingeneral, in the blood glucose meters, each user uses a strip-typebiosensor. An operating principle of such a biosensor is based on anoptical method or an electrochemical method.

The biggest feature of the electrochemical method among these methods isusing an electron transfer mediator. As the electron transfer mediator,ferrocene, derivatives of ferrocene; quinone, derivatives of quinone;transition metal-containing organic and inorganic materials (such ashexaammineruthenium, an osmium-containing polymer, and potassiumferricyanide); and electron transfer organic materials such as anorganic conducting salt and viologen may be used.

A measurement principle of blood glucose using the electrochemicalmethod is as follows. First, the blood glucose is oxidized to gluconatedue to a catalytic action of a glucose oxidase. At this time, FAD, whichis an active site of the glucose oxidase, is reduced and became FADH₂.Then, the reduced FADH₂ is oxidized to FAD through anoxidation-reduction reaction with the electron transfer mediator, andthe electron transfer mediator is reduced. Electrons generated from theelectron transfer mediator in a reduction state formed in this mannerdisperse to an electrode surface. At this time, a concentration of theblood glucose is measured by measuring a current generated by applyingan oxidation potential of the electron transfer mediator in a reductionstate in a working electrode surface.

Unlike a biosensor using a conventional optical method, anelectrochemical biosensor using the above measurement principle mayreduce an influence of oxygen, and even when sample is turbid, it ispossible to use the sample without separate pretreatment.

In addition, a general electrochemical biosensor is made such that anelectrode system including a plurality of electrodes is formed on anelectrically insulating substrate using a method such as screenprinting, and an enzyme reaction layer made of a hydrophilic polymer, anoxidoreductase, and an electron accepter is formed on the formedelectrode system. When a sample solution including a substrate isdropped onto the enzyme reaction layer of the electrochemical biosensor,the enzyme reaction layer is dissolved, the substrate reacts with theenzyme, the substrate is oxidized, and therefore the electron accepteris reduced. After the enzyme reaction is completed, a concentration ofthe substrate in the sample solution may be obtained from an oxidationcurrent that is obtained by electrochemically oxidizing the reducedelectron accepter.

When such a biosensor is used, accurately and rapidly obtaining ameasurement value using a small amount of a sample is a very importantissue in terms of maximizing a user's convenience. Most products formeasuring the blood glucose use a method of sampling blood and thenquantifying the blood glucose in blood using a biosensor. However, sinceblood sampling is a considerably painful operation for the patient, itis necessary to minimize an amount of blood necessary for measurement inorder to reduce the patient's pain. In particular, when 1 μl or less ofa small amount of a sample is used, preferably 0.5 μl or less of asample, and more preferably, 0.3 μl or less of a sample is used, sinceit is possible to sample and measure blood from an alternative site suchas a forearm, it is possible to minimize resulting pain when the patientmeasures the blood glucose. Therefore, the biosensor needs to minimizean amount of blood necessary for measuring the blood glucose.

In addition, another issue of the biosensor is reduced measurementsensitivity according to miniaturization. An amount of areaction-related material fixed to the electrode surface is one of majorfactors influencing sensitivity of the biosensor. However, recently, asthe biosensors are gradually miniaturized, an area to which materials toreact with a specific material may be fixed also decreases. Accordingly,there is a serious limitation of the development of a compact sensorhaving high sensitivity.

SUMMARY

In view of the above-described problems, the present invention providesa reagent composition for a biosensor having high sensitivity, which iscapable of improving analysis linearity of an analyte such as glucoseand minimizing blood necessary for measuring the analyte such asglucose.

The present invention also provides a biosensor, which uses the reagentcomposition and is capable of measuring an analyte such as glucose in anindustrially and economically useful manner.

The above-described and other objects and effects of the presentinvention will be apparent from the following description whichdescribes exemplary examples.

According to an aspect of the present invention, there is provided areagent composition for a biosensor, wherein the reagent compositionincludes at least two kinds of electron transfer mediators includingNaphthol Green B, and an enzyme. Here, the electron transfer mediatormay further include a metal-containing complex.

Preferably, the metal-containing complex may be a ruthenium complex or aferricyanide complex.

Preferably, the ruthenium complex may be at least one selected from thegroup consisting of Ru(NH₃)₆Cl₃,[Ru(2,2′,2″-terpyridine)(1,10-phenanthroline)(OH₂)]²⁺,trans-[Ru(2,2′-bipyridine)₂(OH₂)(OH)]²⁺,[(2,2′-bipyridine)₂(OH)RuORu(OH)(2,2′bpy)₂]⁴⁺, and[Ru(4,4′-bipyridine)(NH₃)₅]²⁺.

Here, the Naphthol Green B equal to or greater than 0.1 parts by weightand equal to or less than 20 parts by weight may be used with respect toa buffer solution being 100 parts by weight.

Here, the enzyme may be any of oxidoreductase, dehydrogenase,transferase, or hydrolase.

Preferably, the oxidoreductase may be at least one selected from thegroup consisting of flavin adenine dinucleotide-glucose dehydrogenase,nicotinamide adenine dinucleotide-glucose dehydrogenase,pyrroloquinoline quinone glucose dehydrogenase, glutamate dehydrogenase,glucose oxidase, cholesterol oxidase, cholesterol esterase, lactateoxidase, ascorbic acid oxidase, alcohol oxidase, alcohol dehydrogenase,and bilirubin oxidase.

Preferably, the reagent composition may further include at least one ofa surfactant, a water-soluble polymer, a fatty acid, a quaternaryammonium salt, and an enzyme stabilizer.

More preferably, the surfactant may be at least one of a nonionicsurfactant, an ampholytic surfactant, a cationic surfactant, an anionicsurfactant, and a natural surfactant.

More preferably, the water-soluble polymer may be at least one of apolyvinyl pyrrolidone (PVP), a polyvinyl alcohol (PVA), perfluorosulfonate, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC),carboxy methyl cellulose (CMC), cellulose acetate, and a polyamide.

Here, the reagent composition may be a reagent composition for a glucosebiosensor.

According to another aspect of the present invention, there is provideda biosensor for measuring an analyte in a sample. The biosensor includesa lower substrate including at least one electrode onto which a reagentcomposition to react with the analyte is applied in a surface; a spacerformed on the lower substrate including a sample injection unit in asurface facing the electrode such that the sample is sucked andintroduced to a position of the electrode; and an upper substrateprovided on the spacer, wherein the reagent composition includes atleast two kinds of electron transfer mediators including Naphthol GreenB, and an enzyme.

Preferably, the electron transfer mediator may further include ametal-containing complex.

Preferably, the enzyme may be any of oxidoreductase, dehydrogenase,transferase, or hydrolase.

Preferably, the metal-containing complex may be a ruthenium complex or aferricyanide complex.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will become more apparent to those of ordinary skill in theart by describing in detail exemplary embodiments thereof with referenceto the accompanying drawings, in which:

FIG. 1 is an exploded perspective view of a biosensor according to anembodiment of the present invention;

FIG. 2 is a glucose measurement current change graph according toComparative Examples 1 to 3 of the present invention;

FIG. 3 is a glucose measurement current change graph according toComparative Examples 4 to 6 of the present invention; and

FIG. 4 is a glucose measurement current change graph according toExamples 1 to 4 of the present invention.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail withreference to examples and drawings of the present invention. Theseexamples are only exemplarily proposed to more specifically describe thepresent invention, and it will be apparent by those skilled in the artthat the scope of the present invention is not limited to theseexamples.

A composition for a biosensor according to an aspect of the presentinvention includes at least two kinds of electron transfer mediatorsincluding Naphthol Green B, and an enzyme.

Naphthol Green B serving as an electron transfer mediator is a sodiumsalt of Naphthol Green Y, has good solubility, is industriallyinexpensive, and is used as a dye in various fields.

Also, the electron transfer mediator includes a metal-containingcomplex.

Here, as the metal-containing complex, materials that may be oxidized orreduced by reacting with enzymes such as a ferricyanide complex,potassium ferricyanide, a ruthenium complex, hexaamminerutheniumchloride, ferrocene and derivatives thereof, quinone and derivativesthereof, phenazine methosulfate and derivatives thereof, p-benzoquinoneand derivatives thereof, 2,6-dichlorophenolindophenol, methylene blue,nitrotetrazolium blue, and an osmium complex, may be used. Among them,as the metal-containing complex, the ruthenium complex or theferricyanide complex is preferably used, more preferably,hexaammineruthenium chloride and potassium ferricyanide may be used, andmost preferably, hexaammineruthenium chloride may be used.

The ruthenium complex in the present invention may be one selected fromthe group consisting of Ru(NH₃)₆Cl₃,[Ru(2,2′,2″-terpyridine)(1,10-phenanthroline)(OH₂)]²⁺,trans-[Ru(2,2′-bipyridine)₂(OH₂)(OH)]²⁺,[(2,2′-bipyridine)₂(OH)RuORu(OH)(2,2′bpy)₂]⁴⁺, and[Ru(4,4′-bipyridine)(NH₃)₅]²⁺. The most preferable ruthenium complex ishexaammineruthenium chloride having a property in that anoxidation-reduction state is stable and reversible in an aqueoussolution, a reduced electron transfer mediator does not react withoxygen, oxidation of the reduced electron transfer mediator isinsensitive to pH, and it has little reaction with an electrochemicalinterfering material such as acetaminophene, ascorbic acid, bilirubin,dopamine, uric acid, and gentisic acid.

In addition, as the enzyme, an oxidoreductase and several kinds ofhydrolases and transferases such as glucose dehydrogenase, glutamateoxaloacetate transaminase (GOT), and glutamate pyruvate transaminase(GPT) may be used.

The oxidoreductase is any of selected from the group consisting offlavin adenine dinucleotide-glucose dehydrogenase, nicotinamide adeninedinucleotide-glucose dehydrogenase, pyrroloquinoline quinone glucosedehydrogenase, glutamate dehydrogenase, glucose oxidase, cholesteroloxidase, cholesterol esterase, lactate oxidase, ascorbic acid oxidase,alcohol oxidase, alcohol dehydrogenase, and bilirubin oxidase.Therefore, when one is selected from among oxidoreductases, a sampleanalyte reacting therewith is selected and a reagent composition isprepared, it is possible to provide a biosensor for detecting any ofglucose, cholesterol, lactate, glutamate, and alcohol.

As will be described below, it can be seen that, in the example of thepresent invention using hexaammineruthenium chloride and Naphthol GreenB together, glucose detection performance significantly increases.

The electron transfer mediator may be appropriately determined bysamples or an oxidoreductase to be used. Also, two kinds or more ofelectron transfer mediators including Naphthol Green B may be combinedand used.

A content of the electron transfer mediator is not specifically limitedbut may be appropriately regulated by an addition amount of the sampleand the like. The electron transfer mediator is preferably prepared as abuffer solution such as glycine.

Meanwhile, the following Reaction Formula represents a measurementprinciple of an electrochemical sensor using an oxidoreductase and anelectron transfer mediator.analyte+enzyme (oxidation state)+electron transfer mediating material(oxidation state) ===>reaction result material+enzyme (oxidationstate)+electron transfer mediating material (reduction state)  [ReactionFormula]

In the Reaction Formula, the electron transfer mediating material in areduction state generated by reacting with an analyte in a sample isproportional to a concentration of the analyte in the sample. Using thisproportion, based on a reference electrode or an auxiliary electrode, aconstant voltage is applied to a working electrode to oxidize theelectron transfer mediating material in a reduction state. A level of anoxidation current generated at this time is measured to quantify of theanalyte in the sample.

As described above, the enzyme reacts with various metabolites to bemeasured and is reduced. Then, the reduced enzyme reacts with theelectron transfer mediator and the metabolite is quantified.

In addition, the reagent composition for a biosensor according to theaspect of the present invention may further include at least one of asurfactant, a water-soluble polymer, a fatty acid, a quaternary ammoniumsalt, and an enzyme stabilizer.

By containing the surfactant, fixing of an oxidoreductase to theelectrode is significantly inhibited and prevented. As a result, it ispossible to improve conversion efficiency from an oxidized form electroncarrier to a reduced form electron carrier due to an oxidoreductase inthe vicinity of the electrode. In other words, it is possible to furtherincrease a correlation with a substrate concentration in the samplesolution. An amount and a form of the surfactant may be selectivelyregulated in order to avoid a degeneration effect in the enzyme.

The surfactant is not particularly limited, as long as an enzymeactivity of the present invention is not decreased. For example, atleast one of a nonionic surfactant, an ampholytic surfactant, a cationicsurfactant, an anionic surfactant, and a natural surfactant may beappropriately selected and used. These may be used alone or in mixturesthereof A preferable surfactant is polyoxyethylene ether. Morepreferably, t-octylphenoxypolyethoxyethanol may be purchased (atrademark: Triton X-100) and used. A concentration of Triton X-100 in areagent mixture is preferably 0.01 to 2 parts by weight with respect to100 parts by weight of a buffer solution.

In addition, the water-soluble polymer among the additives is a polymerscaffold of the reagent composition that stabilizes and disperses theenzyme. As the water-soluble polymer, at least one of a polyvinylpyrrolidone (PVP), a polyvinyl alcohol (PVA), perfluoro sulfonate,hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), cellulose acetate, and a polyamide may be used.

While the present specification describes the biosensor for measuringblood glucose as an example for convenience of description, it is alsopossible to quantify concentrations of various metabolites, for example,biological samples such as cholesterol, lactate, creatinine, protein,hydrogen peroxide, alcohol, amino acid, and enzymes such as glutamatepyruvate transaminase (GPT) and glutamate oxaloacetate transaminase(GOT), and various organic materials or inorganic materials in anenvironmental sample, an agricultural and industrial sample, or a foodsample, using the same method, by introducing an electron transfermediator that is appropriate to a specific enzyme similar to applicationof a blood glucose test.

Therefore, it should be understood that the present invention may beused to quantify various metabolites by changing kinds of enzymesincluded in the reagent composition described in the presentspecification. For example, blood glucose oxidase, lactate oxidase,cholesterol oxidase, glutamate oxidase, horseradish peroxidase, alcoholoxidase, and the like may be used to quantify cholesterol, lactate,glutamate, hydrogen peroxide, and alcohol. An oxidase selected from thegroup consisting of glucose oxidase (GOx), glucose dehydrogenase (GDH),cholesterol oxidase, a cholesterol esterase, lactate oxidase, ascorbicacid oxidase, alcohol oxidase, alcohol dehydrogenase, bilirubin oxidase,and glucose dehydrogenase is preferably used.

Next, an embodiment of a biosensor will be described in detail withreference to FIG. 1, an exploded perspective view of the biosensoraccording to another aspect of the present invention.

The biosensor according to another aspect of the present invention maybe applied to conventional known sensors such as a triglyceride sensorand a cholesterol sensor. The biosensor having a reagent compositionaccording to the present invention is mainly constituted by a lowersubstrate 100, a spacer 200, and an upper substrate 300.

The lower substrate 100 is an insulating substrate in which at least oneof electrodes 21 to 23 are provided on a surface and the above-describedreagent composition that reacts with the analyte in the sample isapplied onto the electrode.

The spacer 200 is formed on the lower substrate 100 and may be boundbetween the lower substrate 100 and the upper substrate 300. A sampleinjection unit 30 may be further provided at a side surface facing theelectrode such that an analyte sample is sucked and induced to theelectrodes 21 to 23 of the lower substrate 100.

The upper substrate 300 is an insulating substrate that faces the lowersubstrate 100, is provided on the spacer 200, and further includes anair discharge unit 40 configured to discharge air that is suckedtogether with the analyte sample through the sample injection unit 30.

In this case, in order to increase a suction rate of the sample, the airdischarge unit 40 may be formed in a tunnel shape in a layer separatefrom the sample injection unit 30. In particular, the air discharge unit40 may preferably have a semi-cylindrical shape that is formed in adirection perpendicular to a suction direction of the sample.

More specifically, the lower substrate 100 may use a thin plate made ofan insulating material such as polyethylene terephthalate (PET),polystyrene, polypropylene, polyimide, an acrylic resin, polyester, PVC,or polycarbonate. The lower substrate 100 may use an insulating materialhaving a thickness of 50 to 400 μm, more preferably, an insulatingmaterial having a thickness of 100 to 300 μm.

Lead wires 24 to 26 in contact with a measurement device (notillustrated) and an electrode system 20 in which the electrodes 21 to 23connected to the lead wires and configured to detect an electricalsignal flowing in the sucked sample are printed are formed on the top ofthe lower substrate 100. Here, the electrodes may include a workingelectrode 23, a reference electrode 22, and a check electrode 21. Thelead wires 24 to 26 may be formed by a method such as general screenprinting. In addition, the electrodes 21 to 23 may be formed by a screenprinting method using conductive carbon ink (carbon paste).

Meanwhile, in order to mutually insulate the electrodes 21 to 23, a partother than some upper surfaces of the electrodes 21 to 23 may bepartially coated with an insulator (insulating paste) to form aninsulating layer. Non-conductive screen printing ink or insulating inkmay be used as such an insulator.

As described above, among a part other than the part in which theinsulator is partially printed, exposed upper surfaces of the electrodes21 to 23 are coated with the reagent composition. The reagentcomposition includes the enzyme to react with the injected sample andthe electron transfer mediator (electron accepter) as described above.The reagent composition is applied onto the electrode and fixed theretoso as to sufficiently cover the electrodes 21 to 23. In addition, thereagent composition may be applied onto all of, at least one of theelectrodes 21 to 23, or only the working electrode 23 and used.

When the sample including the analyte is injected into the reagentcomposition through the sample injection unit 30, the analyte includedin the sample reacts with the enzyme, the analyte is oxidized, andaccordingly the electron accepter is reduced. After an enzyme reactionis completed, an oxidation current obtained by electrochemicallyoxidizing the reduced electron accepter is measured by a measurementdevice (not illustrated) in contact with the lead wires 24 to 26connected to the electrodes 21 to 23. Therefore, it is possible tomeasure a concentration of the analyte included in the sample.

The spacer 200 forms a capillary through the sample injection unit 30that is formed by bonding the upper substrate 300 and the lowersubstrate 100. This allows the analyte sample to be quickly absorbed.

The spacer 200 may be made of a double-sided tape. The sample to bemeasured is automatically injected through the sample injection unit 30due to a capillary phenomenon. Air in the sample injection unit 30 isdischarged externally through the air discharge unit 40 formed in theupper substrate 300 due to introduction of the sample.

Meanwhile, the electrodes 21 to 23 according to the present inventionare manufactured by using a conductive polymer, carbon, ITO particles,metal particles, graphite, and platinum-treated carbon, silver, gold,palladium, or a platinum component. For example, ink made of carbon orplatinum-treated carbon, or palladium-containing ink is used to printthe electrodes 21 to 23 on the lower substrate 100. In addition, theelectrodes 21 to 23 may be formed on the lower substrate 100 by vacuumdeposition using gold.

Hereinafter, configurations of the present invention and resultingeffects will be described in detail with reference to Examples andComparative Examples. However, the present examples are provided todescribe the present invention more specifically, and the scope of thepresent invention is not limited to these Examples.

EXAMPLE 1

Reagent Composition Preparation

pH 6.5 and 50 mM of an ACES buffer solution (ACES:N-(2-acetamido)-2-aminoethanesulfonic acid) was prepared. A polyethyleneglycol being 0.6 parts by weight was put into the prepared buffersolution being 100 parts by weight and dissolved. Then, a surfactantt-octylphenoxypolyethoxyethanol being 0.1 parts by weight (TritonX-100)and an enzyme stabilizer (D-(+)-Trehalose dihydrate) being 1.4 parts byweight were sequentially added and dissolved.

Hexaammineruthenium chloride (Ru(NH₃)₆Cl₃) being 2.3 parts by weight,Naphthol Green B being 0.9 parts by weight, and flavin adeninedinucleotide-glucose dehydrogenase (FAD-GDH) being 0.15 parts by weightwere sequentially added and dissolved in the solution, and thus thereagent composition was prepared.

Biosensor Preparation

A working electrode, a reference electrode, and a check electrode wereformed on a polyester lower substrate by a screen printing method usinga conducting carbon paste. The electrodes were dried for 20 minutes at120° C. using a dry oven, and then an insulating paste was applied usingthe screen printing method.

The working electrode was coated with 1 mg of the reagent compositionand was dried for 20 minutes at 40° C. in the dry oven. A spacer wasattached to the dried lower substrate. Then, an upper substrate wascovered and pressed.

EXAMPLE 2

A reagent composition was prepared as in Example 1. Concentrations ofcompositions other than Naphthol Green B being 1.8 parts by weight werethe same.

A biosensor was prepared the same as in Example 1.

EXAMPLE 3

A reagent composition was prepared as in Example 1. Concentrations ofcompositions other than Naphthol Green B being 3.5 parts by weight werethe same.

A biosensor was prepared the same as in Example 1.

EXAMPLE 4

A reagent composition was prepared as in Example 1. Concentrations ofcompositions other than Naphthol Green B being 6.6 parts by weight werethe same.

A biosensor was prepared the same as in Example 1.

COMPARATIVE EXAMPLE 1

A reagent composition was prepared as in Example 1. However, the reagentcomposition was prepared to have a concentration of hexaamminerutheniumchloride being 1.5 parts by weight, FAD-GDH being 0.15 parts by weight,and having no Naphthol Green B.

A biosensor was prepared the same as in Example 1.

COMPARATIVE EXAMPLE 2

A reagent composition was prepared as in Example 1. However, the reagentcomposition was prepared to have a concentration of hexaamminerutheniumchloride being 3.1 parts by weight, FAD-GDH being 0.15 parts by weight,and having no Naphthol Green B.

A biosensor was prepared the same as in Example 1.

COMPARATIVE EXAMPLE 3

A reagent composition was prepared as in Example 1. However, the reagentcomposition was prepared to have a concentration of hexaamminerutheniumchloride being 4.6 parts by weight, FAD-GDH being 0.15 parts by weight,and having no Naphthol Green B.

A biosensor was prepared the same as in Example 1.

COMPARATIVE EXAMPLE 4

A reagent composition was prepared as in Example 1. However, the reagentcomposition was prepared to have a concentration of Naphthol Green Bbeing 4.4 parts by weight, FAD-GDH being 0.15 parts by weight, andhaving no hexaammineruthenium chloride.

A biosensor was prepared the same as in Example 1.

COMPARATIVE EXAMPLE 5

A reagent composition was prepared as in Example 1. However, the reagentcomposition was prepared to have a concentration of Naphthol Green Bbeing 8.8 parts by weight, FAD-GDH being 0.15 parts by weight, andhaving no hexaammineruthenium chloride.

A biosensor was prepared the same as in Example 1.

COMPARATIVE EXAMPLE 6

A reagent composition was prepared as in Example 1. However, the reagentcomposition was prepared to have a concentration of Naphthol Green Bbeing 13.2 parts by weight, FAD-GDH being 0.15 parts by weight, andhaving no hexaammineruthenium chloride.

A biosensor was prepared the same as in Example 1.

EXPERIMENTAL EXAMPLE

The biosensors prepared by Examples and Comparative Examples were usedto measure a current in a glucose standard solution. Here, the glucosestandard solution refers to blood in which blood taken from veins has42% of hematocrit and has several glucose concentrations using a glucoseanalyzer (manufacturer: YSI Inc., model name: YSI 2300 STAT Plus).

FIG. 2 is a glucose measurement current change graph according toComparative Examples 1 to 3 of the present invention. In FIG. 2, T1represents an experiment result of Comparative Example 1 includinghexaammineruthenium chloride being 1.5 parts by weight and FAD-GDH being0.15 parts by weight. T2 represents an experiment result of ComparativeExample 2 including hexaammineruthenium chloride being 3.1 parts byweight and FAD-GDH being 0.15 parts by weight. T3 represents anexperiment result of Comparative Example 3 including hexaamminerutheniumchloride being 4.6 parts by weight and FAD-GDH being 0.15 parts byweight.

Example experiments were performed using the biosensors according toComparative Examples 1 to 3. As a result, it was determined that thecurrent measured by the glucose concentration did not gradually increaseand linearity was not secured in the biosensor having no Naphthol GreenB among the reagent compositions.

FIG. 3 is a glucose measurement current change graph according toComparative Examples 4 to 6 of the present invention. In FIG. 3, T1represents an experiment result of Comparative Example 4 includingNaphthol Green B being 4.4 parts by weight and FAD-GDH being 0.15 partsby weight. T2 represents an experiment result of Comparative Example 5including Naphthol Green B being 8.8 parts by weight and FAD-GDH being0.15 parts by weight. T3 represents an experiment result of ComparativeExample 6 including Naphthol Green B being 13.2 parts by weight andFAD-GDH being 0.15 parts by weight.

Example experiments were performed using the biosensors according toComparative Examples 4 to 6. As a result, it was determined that thecurrent measured by the glucose concentration did not gradually increaseand linearity was not secured in the biosensor having nohexaammineruthenium chloride among the reagent compositions.

FIG. 4 is a glucose measurement current change graph according toExamples 1 to 4 of the present invention. In FIG. 4, T1 represents anexperiment result of Example 1 including Naphthol Green B being 0.9parts by weight, hexaammineruthenium chloride being 2.3 parts by weight,and FAD-GDH being 0.15 parts by weight. T2 represents an experimentresult of Example 2 including Naphthol Green B being 1.8 parts byweight, hexaammineruthenium chloride being 2.3 parts by weight, andFAD-GDH being 0.15 parts by weight. T3 represents an experiment resultof Example 3 including Naphthol Green B being 3.5 parts by weight,hexaammineruthenium chloride being 2.3 parts by weight, and FAD-GDHbeing 0.15 parts by weight. Finally, T4 represents an experiment resultof Example 4 including Naphthol Green B being 6.6 parts by weight,hexaammineruthenium chloride being 2.3 parts by weight, and FAD-GDHbeing 0.15 parts by weight.

Example experiments were performed using the biosensors according toExamples 1 to 4. As a result, in general, it was determined that thecurrent measured by the glucose concentration gradually increased as theconcentration of Naphthol Green B increases. In addition, it wasdetermined that linearity was improved as the concentration of NaphtholGreen B increases. Particularly, it was determined that the bestlinearity was shown when the concentration of Naphthol Green B was 3.5parts by weight.

In order to induce an appropriate reaction, the reagent compositionaccording to another aspect of the present invention may preferably useNaphthol Green B having a concentration equal to or greater than 0.1parts by weight and equal to or less than 20 parts by weight, and morepreferably use Naphthol Green B having a concentration equal to orgreater than 1 part by weight and equal to or less than 5 parts byweight, with respect to 100 parts by weight of a buffer solution. Thisis because Naphthol Green B having a concentration less than 5 parts byweight may cause an output signal from the reaction to be low, thusdisadvantageously making it impossible to secure linearity, and, on theother hand, Naphthol Green B having a concentration greater than 20parts by weight may lead to a deterioration of solubility of the reagentcomposition, which can cause failure in securing linearity andreproducibility. Most preferably, the reagent composition may useNaphthol Green B having a concentration equal to 3.5 parts by weightwith respect to 100 parts by weight of a buffer solution.

Meanwhile, in the electrochemical biosensor according to the example ofthe present invention, the working electrode, the reference electrode,and the check electrode may be provided on a plane surface of the lowersubstrate. However, the working electrode, the reference electrode, andthe check electrode may also be provided on different surfaces (forexample, an upper substrate) so as to face each other.

According to the present invention, it is possible to improve analysislinearity of an analyte and minimize blood necessary for measuring theblood glucose since a concentration of a small amount of analyte may bedetected.

Since Naphthol Green B of the reagent composition of the biosensoraccording to the present invention has good solubility, it is easy to beindustrially used, and is also cheap. Accordingly, it is possible toprovide a cheap biosensor, which is very useful for people who need tomeasure the blood glucose several times for a day.

However, the effects of the present invention are not limited to theabove-mentioned effects. Other unmentioned effects may be clearlyunderstood by those skilled in the art from the following description.

While detailed examples of the present invention have been specificallydescribed above, it is apparent by those skilled in the art that variousmodifications and alternatives may be made within the scope of thepresent invention, and it may be understood that such modifications andalternatives fall within the appended claims.

What is claimed is:
 1. A biosensor for measuring an analyte in a sample,the biosensor comprising: a lower substrate including at least oneelectrode onto which a reagent composition to react with the analyte isapplied on a surface of the at least one electrode; a spacer formed onthe lower substrate including a sample injection unit facing the atleast one electrode such that the sample is sucked and introduced to aposition on the at least one electrode; and an upper substrate providedon the spacer, wherein the reagent composition includes at least twokinds of electron transfer mediators including Naphthol Green B, and anenzyme, and wherein the Naphthol Green B equal to or greater than 1parts by weight and equal to or less than 5 parts by weight is used withrespect to a buffer solution being 100 parts by weight.
 2. The biosensoraccording to claim 1, wherein the electron transfer mediators furtherinclude a metal-containing complex.
 3. The biosensor according to claim1, wherein the upper substrate further includes an air discharge unitconfigured to discharge air in the sample.
 4. The biosensor according toclaim 3, wherein the air discharge unit has a semi-cylindrical shapethat is formed in a direction perpendicular to a suction direction ofthe sample.
 5. The biosensor according to claim 1, wherein the enzyme isat least one selected from the group consisting of oxidoreductase,dehydrogenase, transferase, and hydrolase.
 6. The biosensor according toclaim 2, wherein the metal-containing complex is a ruthenium complex ora ferricyanide complex.